Stable crystal modifications of DOTAP chloride

ABSTRACT

The invention relates to enantiomerically pure DOTAP chloride and stable crystal modifications of (2R,S)-, (2S)- and (2R)-DOTAP chloride, to a process for the preparation of these modifications, and to the use thereof as constituent for the preparation of medicaments.

The present invention relates to enantiomerically pure DOTAP chlorideand crystal modifications of racemic and enantiomerically pure DOTAPchloride, to a process for the preparation thereof, and to the usethereof for the preparation of pharmaceutical compositions.

DOTAP chloride above and below denotesN,N,N-trimethyl-2,3-bis[[(9Z)-1-oxo-9-octadecenyl]oxy]-1-propanaminiumchloride, also known as (Z,Z)—N, N,N-trimethyl-2,3-bis[(1-oxo-9-octadecenyl)oxy]-1-propanaminium chlorideor 1,2-dioleoyloxy-3-trimethylammonium propane chloride, and thehydrates thereof.

CAS numbers: 132172-61-3 and 477274-39-8 (racemate),197974-73-58 (racemate, monohydrate)428506-51-8 (2S form), 328250-28-8 (2R form)

Liposomes are synthetic multilayered vesicles (sphericallyself-contained membranes) comprising ambiphilic substances, usuallynatural lipids, into which both hydrophilic substances can beencapsulated into the aqueous interior, and also lipophilic substancescan be incorporated into the inside of the lipid membrane.

They are employed in particular in cosmetics and in medicine, especiallyin dermatology. Here, in particular vitamins, coenzymes, skin-careagents and sunscreens are embedded. Liposomes are generally appliedtopically.

However, liposomes are increasingly achieving further importance inpharmaceutical technology, since parenteral application of liposomesenables more specific organ distribution to be achieved than if theactive compounds are used in freely dissolved form.

If DNA, RNA or proteins are incorporated, lipoplexes are obtained.

Addition of oils and the use of high-pressure homogenisers enables theformation of so-called nanoparticles (nanoparts) to be forced fromliposomes. These are particles of approximately the same size asliposomes, but which do not have a water phase, but instead an oil phasein their interior. They are particularly suitable for the encapsulationof lipophilic substances.

Microemulsions are colloidally disperse, single-phase systems comprisingaqueous, lipid-like and surfactant components. They have a particle sizeof 1-500 nm and behave in a similar manner to liquids.

Especially in connection with peptidic active compounds, nucleotides,vaccines and other biopharmaceuticals, which normally have poorsolubility, the solubilising effect has very great importance in thecase of the applications described above.

In addition, degradation of the active compounds in the body can beslowed and a sustained-release effect achieved in this way.

DOTAP chloride belongs to the class of cationic lipids. In contrast tonaturally occurring phospholipids, these do not have a zwitterioniccharacter. Liposomes comprising cationic lipids, alone or combined withphospholipids or other lipid-like compounds, have a positively chargedsurface. This gives rise to high affinity to cells which have anegatively charged surface on the outside, for example endothelialcells.

Particularly important, however, is the ability of DOTAP-based and othercationic liposomes and lipoplexes to penetrate into cells and thus totransport the active compounds incorporated therein into the interior ofthe cell (transfection).

All these properties make DOTAP chloride very interesting for cancertherapy too. These properties give rise to the possibility of applyingconventional cytostatic agents incorporated in cationic DOTAP liposomes.

The transfection properties of DOTAP chloride and other DOTAP salts,such as, for example, the acetate, bromide, dihydrogenphosphate,hydrogensulfate, iodide, mesylate, methylsulfate, trifluoroacetate,sulfate or disulfate and triflate, are adequately known from theliterature.

DOTAP dihydrogenphosphate and DOTAP mesylate are only mentioned asracemate in the literature. All other salts mentioned above are eachmentioned as racemate and as 2S-enantiomer, and in addition the2R-enantiomers of the chloride and methylsulfate are mentioned.

In some in-vitro studies, other salts, such as, for example, DOTAPmethylsulfate, have achieved better transfection rates than DOTAPchloride.

Used in vivo, however, anion exchange at the liposome surface takesplace in the living body, meaning that the advantages of other salts donot arise here. Especially on medical use in humans in particular forparenteral application, DOTAP salts with physiologically acceptableanions, such as, for example, the corresponding chloride or the acetate,are therefore preferred.

Medical, in particular parenteral applications make the highest demandsof the quality and purity of the active compounds and adjuvants used.There are therefore very strict regulations on the part of theauthorities with respect to the preparation, reproducibility ofpreparation and by-product profile of these compounds. In the case ofsubstances used parenterally, microbiological contamination bypathogenic microorganisms and endotoxins must, in addition, be strictlyavoided and controlled.

DOTAP chloride and other DOTAP salts are extremely unstable and aretherefore difficult per se to prepare in an acceptable purity so thatthey are suitable for use for the preparation of a medicamentformulation.

Like all lipids which carry oleic acid radicals, such as, for example,the natural phospholipids DOPC and DOPE, all DOTAP salts are verysensitive to oxidation. However, the oxidation products of unsaturatedfatty acid derivatives generally have high toxicity.

Suitable preparation and purification methods are required here. DOTAPacetate, for example, is in the form of a high-boiling oil andindustrially can therefore only be obtained with great difficulty inadequate quality.

The conventional methods of overcoming the instability, such as, forexample, the addition of antioxidants in the form of ascorbic acid orreduced L-glutathione, greatly restrict the general usability of DOTAPchloride since interactions with the active compounds to be embeddedlater cannot be excluded. Complete exclusion of oxygen during thepreparation, storage and use is virtually impossible or can only befacilitated with very great effort.

DOTAP chloride is commercially available only as a chloroform solutionor as an amorphous solid.

In addition to its oxidation sensitivity, amorphous DOTAP chloride isalso extremely hygroscopic and deliquesces within an extremely shorttime at normal atmospheric humidity levels to give a greasy film. Thismakes handling of this compound much more difficult.

Thus, the manufacturer of amorphous DOTAP chloride generally recommendsstorage under protective gas at −20° C. and only guarantees a shelf lifeof about 6 months.

The literature only reveals various synthetic routes for the preparationof amorphous, racemic DOTAP chloride:

Eibel and Unger, DE4013632A1, outline the synthesis of DOTAP chloridefrom DOTAP bromide by ion exchange in the chloroform/methanol/aqueousHCl solvent system followed by purification by means of chromatography.DOTAP bromide is obtained in advance in situ from1-bromo-2,3-dioleoyloxypropane.

Leventis and Silvius, Biochim. Biophys. Acta, 1023 (1990) 124-132,report on the synthesis of DOTAP chloride from DOTAP iodide by ionexchange in the two-phase solvent/NaCl solution system. DOTAP iodide isobtained in advance by methylation of the corresponding dimethylaminocompound by means of methyl iodide.

Nantz et al., Biochim. Biophys. Acta, 1299 (1996) 281-283, J. Med. Chem.40 (1997) 4069-4078, describe the synthesis of DOTAP chloride bynon-aqueous ion exchanger chromatography. The desired compound isobtained by evaporation of the eluate.

Felgner et al., U.S. Pat. No. 5,264,618, carry out the methylation ofthe corresponding dimethylamino compound directly to DOTAP chloride bymeans of methyl chloride. They apparently obtain a yellow wax bycrystallisation from acetonitrile at −20° C. However, DOTAP chloride isvirtually insoluble in acetonitrile at room temperature. Attempts toreproduce this so-called crystallisation gave only amorphous materialthrough solidification of the oily substance obtained from hot solutionon cooling. The fact that this is not a crystallisation is also evidentfrom the fact that the authors apparently do not achieve a purificationeffect and have to purify the substance by chromatography.

Consequently, neither synthetic routes for the preparation of the twoenantiomeric DOTAP chlorides nor characteristic properties thereof areknown to date. Although Chemical Abstracts has assigned numbers for thetwo enantiomers, the corresponding publications describe exclusivelywork with racemic DOTAP chloride.

In particular if the compounds are intended for parenteral use, apreparation which includes treatment with ion exchanger resin isextremely problematical in view of possible microbiologicalcontamination, since corresponding resins are an ideal nutrient mediumfor bacteria and even after they have been killed, a risk ofcontamination by endotoxins still remains.

The object of the present invention is therefore to provide DOTAPchloride salts and hydrates in high purity and with adequate chemicaland physical stability. A further object of the present invention is toprovide these salts with long shelf lives, enabling them to be used forthe preparation of pharmaceutical formulations. There continues to be agreat demand for a reproducible process for the preparation of stableforms of DOTAP chloride salts and hydrates which can be carried out onan industrial scale.

Enantiomerically pure DOTAP chloride can be obtained fromenantiomerically pure starting materials analogously to the processesdescribed for the racemate, i.e.

via (R or S)-1-chloro-2,3-dioleoyloxypropane,

via (R or S)-1-LG-2,3-dioleoyloxypropane and ion exchange (LG=leavinggroup) or via (R or S)-1-dimethylamino-2,3-dioleoyloxypropane.

A further preparation method which may be mentioned is racemateresolution of racemic DOTAP chloride.

By means of experiments, it has now been found, surprisingly, that bothracemic and also enantiomerically pure, crystalline DOTAP chloride canbe obtained in a simple manner with high chemical purity and excellentstability. The crystalline products obtained in this way have virtuallyunlimited stability at room temperature under protective gas. They aretherefore suitable as constituent or as starting material for thepreparation of medicament forms.

The present invention accordingly relates to enantiomerically pure DOTAPchloride and stable crystal modifications of racemic andenantiomerically pure DOTAP chloride.

The stable crystal modifications can be in crystalline and partiallycrystalline form. They have a never hitherto achieved purity of >98%together with a never hitherto achieved stability of >98% in relation tothe starting value after storage for 12 months with exclusion of air at25° C. and 60% relative atmospheric humidity. (see in this respect Table1). The DOTAP chloride crystal modifications have a content of less than1 equivalent of water or solvent of crystallisation per equivalent ofDOTAP chloride.

The racemic DOTAP chloride crystal modifications exist, for example, inthree different crystal modifications (type I, type II, and type III)and exhibit moderately sharp bands in powder X-ray diffractionmeasurements (see in this respect FIG. 1 to FIG. 3 and Table 2).

Selected 2 theta values for the various crystal modifications are 12.6,19.5, 20.2, 21.5 and 25.2 (type 1); or 3.3, 4.9, 19.3, 20.0 and 23.5(type II); or 2.8, 5.8, 20.0, 21.2 and 25.1 (type III).

Enantiomerically pure DOTAP chlorides are likewise obtained incrystalline form. Selected 2 theta values for the crystal modificationfound are 12.8, 19.4, 19.8, 20.2, and 21.5 (type IV, see in this respectFIG. 4).

The enantiomers are optically active. Thus, (2S)-DOTAP chloride has anoptical rotation of −2.12°, (2R)-DOTAP chloride has an optical rotationof +2.12° ([α]_(D) at 20° C., 1% solution in dichloromethane).

The invention furthermore relates to a process for the preparation ofDOTAP chloride crystal modifications which is characterised in thatDOTAP chloride is crystallised from an aprotic medium. The aproticmedium used for this purpose can be aprotic solvents or mixturesthereof.

The aprotic medium may also comprise protic solvents, such as, forexample, water, in small amounts. In exceptional cases, 25% by weight ofprotic solvents may also be present under suitable conditions. Thecrystallisation of the DOTAP chlorides can be carried out here directlyfrom the reaction solution without prior purification. Likewise,crystalline DOTAP chloride can be obtained by recrystallisation ofamorphous, partially crystalline or crystalline material.

Suitable aprotic solvents are, in particular,

-   ethers, such as, for example, tetrahydrofuran,    methyltetrahydrofuran, dioxane, diethyl ether, dipropyl ether,-   diisopropyl ether and methyl tert-butyl ether,-   ketones, such as, for example, acetone and 2-butanone, methyl    isobutyl ketone, methyl isopropyl ketone,-   nitriles, such as, for example, acetonitrile, and-   esters, such as, for example, ethyl formate, methyl acetate, ethyl    acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl    acetate, dimethyl carbonate, diethyl carbonate and    1,3-di-oxolidin-2-one.

These solvents may in each case be used in pure form or in the form of amixture, i.e. it is possible both to use the various aprotic solvents ina group in the form of a mixture and also to employ aprotic solventtypes inn the form of a mixture with one another. As already indicatedabove, protic solvent additions may be present in the aprotic solvent orsolvent mixture used.

Protic solvent additions of this type can typically consist of thefollowing solvents:

-   alcohols, such as, for example, methanol, ethanol, n-propanol,    isopropanol, n-butanol, isobutanol, 2-butanol, tert-butanol,    3-methyl-1-butanol and ethylene glycol, methoxyethanol,    ethoxyethanol, or    water.

The protic solvent additions may in turn be additions of the puresolvents or of mixtures of these protic solvents.

The crystallisation of the DOTAP chloride modifications is generallyachieved specifically by slow cooling of the prepared solution totemperatures below 30° C. The formation of the crystals is carried outeither spontaneously or by inoculation with the corresponding DOTAPchloride crystal modification.

The various DOTAP chloride crystal modifications can be converted intoone another. The conversions can be achieved by heat treatments of theisolated crystal modifications at elevated temperature or by extendedstirring of their suspensions under crystallisation conditions.

The use of amorphous or partially crystalline DOTAP chloride as startingmaterial for the recrystallisation gives, by the process described,essentially crystalline DOTAP chlorides of never hitherto achievedpurity together with never hitherto achieved stability.

The invention also relates to the use of crystalline DOTAP chlorides forthe preparation of medicament formulations since the crystalline DOTAPchlorides have excellent stability in solid form under the statedconditions and have constant and very good quality for a virtuallyunlimited time.

In addition, enantiomerically pure DOTAP chlorides have differentphysical properties to the racemate, in particular in combination withchiral compounds, such as the phospholipids or cholesterol.

The novel properties found for the enantiomerically pure DOTAP chloridescan advantageously be utilised, alone or in combination with suitablephospholipids, cholesterol and derivatives thereof, to provide novelliposome grades which, compared with conventional forms, on the one handrepresent closer packings of the lipids and on the other hand have amore uniform structure. Thus, liposomes prepared from the pureenantiomers exhibit a 5° C. higher principal phase transitiontemperature compared with the liposomes comprising racemic DOTAPchloride. This is a measure of the packing density. Liposomes comprisingDOTAP chloride enantiomers therefore also have reduced leaking of thecompound incorporated in them.

This has the consequence that liposomes charged with pharmaceuticallyactive compounds will release active compounds in a delayed manner ininteraction with the metabolism in the human or animal body. Inparticular, sensitive active compounds can thus advantageously betransported in a more specific manner to the desired site or organ wherethe medicament action is desired.

The DOTAP chloride enantiomers also exhibit, in particular incombination with chiral lipids, such as phospholipids, cholesterol andderivatives thereof, transfection properties which differcell-line-specifically.

For the preparation of these novel liposomes, it is possible for theperson skilled in the art specifically to select a form or a certainmixing ratio of the DOTAP chlorides thus provided in order to prepareliposomes having certain novel properties.

The invention consequently furthermore also relates to thepharmaceutical compositions resulting from the use of the DOTAP chlorideforms claimed. Pharmaceutical compositions of this type can comprise thecrystal modifications of (2R,S)-, (2S)- and (2R)-DOTAP chloride togetherwith other pharmaceutical active compounds and known adjuvants usuallyemployed in medicament preparation, as well as one or more solvents.

These pharmaceutical compositions can, for example, be in the form ofliposomes, lipoplexes, microemulsions and nanoparticles and include, forexample, an active compound from the group of the peptides, nucleotides,vaccines or cytostatic agents.

The present description enables the person skilled in the art to applythe invention in a comprehensive manner. In addition, the followingexamples serve for better understanding and for illustration of possiblevariants of the invention. These examples should therefore in no way beregarded as restrictive.

All temperatures mentioned in the following examples are indicated indegrees Celsius. Unless stated otherwise, content data are given as % byweight.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates x-ray spectra for DOTAP chloride crystalline Type I,

FIG. 2 illustrates x-ray spectra for DOTAP chloride crystalline Type II,

FIG. 3 illustrates x-ray spectra for DOTAP chloride crystalline TypeIII,

FIG. 4 illustrates x-ray spectra for DOTAP chloride crystalline Type IV,

FIG. 5 illustrates x-ray spectra for amorphous DOTAP chloride, and

FIG. 6 illustrates DSC curves for (2RS)-, (2R)- and (2S)-DOTAPchlorides.

X-RAY SCANNING INFORMATION REGARDING THE FIGURES

FIG. 1

Comment: Single Measurement-Hygroscopic

Diffract.: Transmission

Monochrome: Curved Germanium (111)

Radiation: 1.54060 Cu

Generator: 55 kV, 30 mA

Detector: Linear PSD/Moving/Fixed Omega

Scan Mode: Transmission

Range 1: 2Theta (begin, end, step)=2.000, 49.980, 0.020 130.0 sec/stepImax=7060 (PSD Step 0.50 10.0 sec/step)

FIG. 2

Diffract.: Transmission

Monochrome: Curved Germanium (111)

Radiation: 1.54060 Cu

Generator: 55 kV, 30 mA

Detector: Linear PSD/Moving/Fixed Omega

Scan Mode: Transmission

Range 1: 2Theta (begin, end, step)=2.000, 49.980, 0.020 130.0 sec/stepImax=4708 (PSD Step 0.50 10.0 sec/step)

FIG. 3

Comment: Single Measurement

Diffract.: Transmission

Monochrome: Curved Germanium (111)

Radiation: 1.54060 Cu

Generator: 55 kV, 30 mA

Detector: Linear PSD/Moving/Fixed Omega

Scan Mode: Transmission

Range 1: 2Theta (begin, end, step)=2.000, 49.980, 0.020 130.0 sec/stepImax=1871 (PSD Step 0.50 10.0 sec/step)

FIG. 4

Diffract.: Transmission

Monochrome: Curved Germanium (111)

Radiation: 1.54060 Cu

Generator: 55 kV, 30 mA

Detector: Linear PSD/Moving/Fixed Omega

Scan Mode Debye-Scherrer

Range 1: 2Theta (begin, end, step)=2.000, 49.980, 0.020 130.0 sec/stepImax=2995 (PSD Step 0.50 10.0 sec/step)

FIG. 5

Diffract.: Transmission

Monochrome: Curved Germanium (111)

Radiation: 1.54060 Cu

Generator: 55 kV, 30 mA

Detector: Linear PSD/Moving/Fixed Omega

Scan Mode Debye-Scherrer

Range 1: 2Theta (begin, end, step)=2.000, 49.980, 0.020 120.0 sec/stepImax=2102 (PSD Step 0.50 10.0 sec/step)

EXAMPLES FOR ILLUSTRATING THE INVENTION Example 1 Stabilities

In order to determine the stability of crystalline DOTAP chlorides, thesubstances are stored together with comparative samples at 25° C. and60% relative humidity with exclusion of air. The remaining content ofDOTAP chloride is measured at periodic intervals and quoted incomparison to the initial value.

The purity and content of DOTAP chloride are determined by means ofHPLC. For type I, the following values are found:

The stability determination can be repeated at any desired time, thevalues indicated in Table 1 are reproducible.

TABLE 1 (R,S)-DOTAP chloride crystalline Exposure time in months type I0 1 2 3 6 12 Area-%  100% 100.0% 100.0% 100.0% 100.0% 100.0% % by weight98.6%  97.%  97.9%  97.2%  98.2%  98.7%

Example 2 Powder X-ray Diagrams

For characterisation of the structural properties (crystalmodifications) of crystalline DOTAP chlorides, powder X-ray diagrams(diffraction spectra) of these substances are recorded.

Crystalline DOTAP chlorides give spectra with moderately sharp bandswhich have relatively good resolution for lipids. The spectra indicatehigh crystalline contents. No amorphous fractions are visible under thepolarising microscope.

Examples of spectra are shown in FIG. 1 (type I), FIG. 2 (type II), FIG.3 (type III) and FIG. 4 (type IV).

For comparison, a spectrum of a commercially available, amorphous sampleis shown in FIG. 5 (amorphous).

Table 2 lists selected 2 theta values for the various crystalmodifications of racemic and enantiomerically pure DOTAP chlorides:

TABLE 2 Type Selected 2 theta values Type I racemic 12.6, 19.5, 20.2,21.5 and 25.2 Type II 3.3, 4.9, 19.3, 20.0 and 23.5 Type III 2.8, 5.8,20.0, 21.2 and 25.1 Type IV enantiomerically 12.8, 19.4, 19.8, 20.2, and21.5 pure

Example 3 Principal Phase Transition Temperatures

Differential scanning caliometry (DSC) measurements are carried out onmultilamellar liposomes in water. Liposomes are prepared by thethin-film method from the calculated amounts of racemic orenantiomerically pure DOTAP chloride. The lipid concentration here is ineach case 0.1 g/ml. Suitable amounts of these dispersions are thenintroduced into sealable aluminium crucibles and measured using a 204Phoenix calorimeter (Netzsch, Selb, Germany). In each case, threesuccessive heating/cooling runs from −50° C. to +20° C. are carried outat 1° C./min.

For all three DOTAP chloride variants, phase transition temperaturesbelow 0° C. are found. For the cooling cycles, this is in each case at−23° C. to −24° C. Differences between racemic and enantiomerically pureDOTAP chloride are in each case evident in the heating cycles.(2R)-DOTAP chloride and (2S)-DOTAP both exhibit an endothermic phasetransition around −12.5° C., while the phase transition for the racemateis at −17.5° C. (see FIG. 6).

Example 4 Transfection Properties on COS-7 Cells

Racemic and enantiomerically pure DOTAP chloride and racemic DOTAPmethylsulfate are each dispersed with the same amount of cholesterol inthe transfection medium and treated with ultrasound. The liposomes andGFP plasmid solution are mixed and incubated for 15 minutes, amounts per6 well: 2 μg of plasmid/8 μg of lipid.

After incubation for 5 hrs, the supernatant is removed from the cells bysuction at 37° C./5% CO2, 2 ml of fresh medium is added, and the mixtureis incubated for a further 20 hours. After work-up, FACS analysis showsefficient transfection for all lipoplexes. A significant differencebetween the transfection rates for the individual lipid mixtures isevident:

(R)-DOTAP chloride/cholesterol complex: 32.4% (S)-DOTAPchloride/cholesterol complex: 11.0% (R,S)-DOTAP chloride/cholesterolcomplex: 25.9% (R,S)-DOTAP methylsulfate/cholesterol complex: 20.2%

The invention claimed is:
 1. A pharmaceutical composition comprisingenantiomerically pure (2R)-DOTAP chloride and a pharmaceuticallyacceptable carrier, with the proviso that the composition issubstantially free of (2S)-DOTAP chloride.
 2. A pharmaceuticalcomposition according to claim 1, further comprising one or morepeptides, nucleotides, vaccines or cytostatic agents, or a mixturethereof.
 3. A pharmaceutical composition according to claim 1, furthercomprising one or more liposomes, lipoplexes, nanoparticles ormicroemulsions, or a mixture thereof.